GABA is the major inhibitory neurotransmitter in the mammalian brain, and dysfunction of the GABAergic system underlies a number of neurological and neuropsychiatric disorders such as depression, schizophrenia and epilepsy. Currently 1H MRS is the only noninvasive method to measure brain GABA in humans. Altered GABA levels detected by MRS occur in a variety of clinical conditions such as depression and epilepsy, and recent reports indicate a strong relationship between GABA concentration and cortical excitability (as assessed by fMRI and gamma EEG) and cortical interconnectivity (as assessed by EEG and resting state fMRI). While the GABA MRS measurement has gained increasing importance as a biomarker in both clinical and basic neuroscience research, key questions about its meaning remain: ? Does the GABA MRS measurement provide an accurate assessment of total brain GABA? ? Does cytoplasmic GABA, which is most likely the primary component of the GABA MRS signal, determine the concentration of extracellular fluid (ECF) GABA, and therefore act as an important mechanism for tonic extrasynaptic GABA inhibition? ? Can the cytoplasmic and vesicular subcomponents of the GABA MRS signal be separately quantitated? ? What is the relation between physiological changes in cellular and extracellular GABA concentration and changes in cortical excitability? We propose to address these questions in a well-established rodent model that allows MRS, fMRI, microdialysis, and electrophysiology to be performed in parallel. In Aim 1 the in vivo MRS visibility of GABA will be determined, and whether the 1H MRS GABA signal can act as a biomarker of extracellular fluid GABA and tonic inhibition, through measurement of ECF GABA, cellular GABA, and cortical excitability. In Aim 2 a novel diffusion-weighted MRS method will be employed to characterize the cytoplasmic and vesicular components to the 1H MRS GABA signal to allow the two components of the GABA inhibition system to be studied in vivo. These aims will leverage our extensive experience and advances in 1H MRS GABA measurements with our strong collaborations with clinical neuroscientists who will provide expertise in microdialysis, electrica recordings, and pharmacological and functional manipulations of the GABAergic system. Successful completion will provide critical methodological validation of the MRS GABA measurement, develop a novel method to differentiate cytoplasmic and vesicular GABA pools, and gain new insight into how physiological and pharmacologically-induced shifts in GABA levels relates to GABA inhibitory function, and thus the meaning of [GABA]MRS as a translational biomarker.